Isozyme electrophoresis protocols

DAVID C. TAYLOR stddct at UNX1.SHSU.EDU
Mon Mar 10 10:44:34 CST 1997


I am interested in a discussion about isozyme electrophoresis protocol
development for Pteridophytes.

As this is not the most appropo forum for this discussion, you may please
just hit delete now.  If you know a better forum or source of
information, please let me know. If you can help to answer my questions,
please respond 'off-list' to my e-mail account.  Thanks for your patience
:-).

Specifically, I am ready to run a series of isozymes on starch gels of
the fern Thelypteris sp.  I have adapted protocols primarily from:

Soltis, D. E., et al., 1983. Starch Gel Electrophoresis of Ferns: A
        Compilation of Grinding Buffers, Gel and Electrode Buffers, and
        Staining Schedules.  Am. Fern J. 73(1): 9-27.

This paper is very thorough in its treatment of techniques to minimize
the inactivation of secondary compounds prevalent in ferns.  However,
there is no mention of gel running times or voltage/current settings.

Having previously run some Allium gels, I decided to start with the
protocols I used in that study:  start at 200V (current about 25mA) for
1h removing the wicks after 15 min, increasing the voltage to 300V for 3.5h.

Initially, I noticed that 200V caused the gels to run at about 50mA,
which seems high, condsidering I do not want to burn up the enzymes or
run them off the gel.  I used a bromphenol blue dye front, and stopped
the gels after the prescribed time.  Staining for phosphoglucoisomerase
(PGI) produced bands that were smears from the starting line to where
they wound up.  I was encouraged that I did get staining in all lanes, as
I was testing 3 separate grinding buffers, and considering the relatively
high (?) current.

My question then is how does one go about modifying the various variables
(pH, starch concentration, time, voltage, current, did I miss any?) to
get the discrete bands necessary for scoring the zymograms?

I hope this question is not too naive.

Thanks in advance for any help in this matter.

Sincerely,

David C. Taylor, Undergraduate Student
Department of Biological Sciences, Sam Houston State University
GIS Analyst, Texas Regional Institute for Environmental Studies
stddct at unx1.shsu.edu             http://www.shsu.edu/~stddct/




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